1. Field of the Invention
This invention relates to an antibody which recognizes a human pre-B cell receptor. The invention also relates to an antibody which reacts with a VpreB molecule of a surrogate light chain of a human pre-B cell receptor expressed on the surface of a human pre-B cell, VpreB in the cytoplasm of the pre-B cell, and a VpreB molecule expressed in the cytoplasm a human pro-B cell; or an active fragment of this antibody. The invention further relates to a method of detecting a human pro-B cell and a human pre-B cell by the above-mentioned antibody or its active fragment with the use of a saponin buffer. The antibody of the present invention can be applied to the diagnosis of acute lymphoblastic leukemia.
2. Related Background Art
B cell is known to differentiate from a hematopoietic stem cell of bone marrow into a plasma cell through the stages of pro-B cell, pre-B cell, immature B cell, and mature B cell.
On the surface of a B cell, immunoglobulin is expressed (may be referred to hereinafter as Ig) as an antigen-recognizing receptor. The antigen-recognizing receptor is composed of two types of polypeptides, i.e., a heavy chain and a light chain. Its composition varies as the B cell is differentiated.
Differentiation of B cell and its antigen-recognizing receptor will be described with a mouse taken as an example.
In pro-B cell, it is known that a surrogate light chain (SL chain), which is composed of two associated polypeptides called xcex5 and vpreB, is associated with a heavy chain (H chain) called a surrogate heavy chain (SH chain), and the associated pair is expressed on the cell surface. The surrogate light chain is so called, because the xcex5 molecule (may be referred to hereinafter as xcex5) and the VpreB molecule (may be referred to hereinafter as VpreB) are associated together to substitute for an ordinary light chain (L chain). xcex5 corresponds to the constant region of a light chain, while VpreB corresponds to the variable region of the light chain.
With regard to pre-B cell, a pre-B cell receptor is known to be expressed on the surface of the cell. In the pre-B cell receptor, a surrogate light chain (SL chain), which is composed of two associated polypeptides called xcex5 and VpreB, is associated with a heavy chain (H chain) called xcexc chain.
In an immature B cell and a mature B cell, a B cell receptor composed of an ordinary L chain bound to xcexc chain is expressed as an antigen-recognizing receptor.
As will be seen from the foregoing description, xcex5 and VpreB are expressed only in pro-B cell and pre-B cell.
With a human pre-B cell receptor, similar findings have been reported.
The following are known as antibodies which react with the human pre-B cell receptor. Their reactivity, etc. are shown in Table 1.
{circle around (1)} Anti-human pre-B cell receptor antibodies: SLC1, SLC2, SLC3, SLC4 (Cell, Vol. 73, 73-86, 1993)
{circle around (2)} Anti-human pre-B cell receptor antibody: 9C2 (Eur. J. Immunolo., Vol. 24, 257-264, 1994)
{circle around (3)} Anti-human VpreB antibodies: 3C7, 6F6 (Eur. J. Immunolo., Vol. 26, 2172-2180, 1996)
{circle around (4)} Anti-human VpreB antibodies: B-MAD176, B-MAD688, B-MAD792, B-MAD1112 (J. Exp. Med., Vol. 183, 2693-2698, 1996)
An abnormality in B cell differentiation causes leukemia. Knowing at which stage of differentiation B cell multiplied abnormally is important in treating leukemia. In acute lymphoblastic leukemia in childhood, in particular, abnormal proliferation of pro-B cell or pre-B cell may be a frequent cause. Currently, B cell surface markers generally used in diagnosis are CD10, CD19, CD20, and CD21. Attempts have been made to react any of these antibodies with B cell and to determine by the results which stage the B cell is at. However, CD19, CD20, and CD21 also react with an immature B cell or a mature B cell, while CD10 also reacts with a T cell or an epithelial cell. These antibodies, therefore, were unable to detect only a human pro-B cell or a human pre-B cell specifically.
The present invention aims to provide an antibody which recognizes a human pre-B cell receptor, and which does not recognize a human pro-B cell, thereby making it possible to distinguish between human pre-B cell and human pro-B cell and detect human pre-B cell or human pro-B cell specifically.
In the field of clinical diagnosis, microscopic observation of a cell is made. Compared with other methods, this is the most convenient and fastest method that requires the smallest space for installation of equipment. If staining of the cell is very weak, however, it cannot be observed under a microscope, although it can be detected by a highly accurate detection method such as FACS. All the antibodies against a human pre-B cell receptor enumerated earlier as above-mentioned {circle around (1)} to {circle around (4)}, especially the antibodies against VpreB, only gave the results with FACS. That is, it has not been shown whether they have reactivity enough to stain the antibody-reacted cell so as to be observable microscopically. Also, it has been shown that reactivity with a human pro-B cell is only reactivity on the surface of the cell, and intracellular staining has not been shown.
It is another object of the present invention to provide an antibody which reacts specifically with a human pre-B cell and a human pro-B cell, and which can immunostain a human pre-B cell expressing a human pre-B cell receptor on the surface and a human pro-B cell expressing a VpreB molecule intracellularly so that only the immunostained cell can be observed microscopically.
To attain this object, the present invention provides an antibody which recognizes a human pre-B cell receptor and does not recognize a human pro-B cell. The antibodies of this invention are an antibody which recognizes a stereostructure formed by the xcexc chain, xcex5 and VpreB of a human pre-B cell, and an antibody which reacts with a VpreB molecule, one of the constituents of the SL chain of a human pre-B cell receptor expressed on the surface of a human pre-B cell, or reacts with a VpreB molecule expressed in the cytoplasm of a human pro-B cell, but does not react with a xcex5 molecule, and whose reactivity is such that the human pre-B cell or human pro-B cell reacted with the antibody can be detected microscopically.
The invention also provides an anti-human pre-B cell receptor antibody as claimed in claim 1 which is a monoclonal antibody produced by hybridoma HSL2 (originally deposited on Oct. 16, 1997 under acceptance No. FERM-P16476 with National Institute of Bioscience and Human Technology (NIBHT), Agency of Industrial Science and Technology, Ministry of Industrial Trade and Industry, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan; and transferred on May 25, 1998 under acceptance No. FERM-BP6378 to NIBHT, the international deposition organization), or an active fragment of the monoclonal antibody.
The invention also provides an anti-human pre-B cell antibody or its active fragment which is a monoclonal antibody produced by hybridoma HSL96 (originally deposited with NIBHT on May 30, 1997 under acceptance No. FERM-P16251; and transferred on May 25, 1998 under acceptance No. FERM-BP6375 to NIBHT, the international deposition organization), or an active fragment of the monoclonal antibody.
The invention also provides a method for detecting a human pro-B cell, which comprises immunostaining a human pro-B cell in a sample with the use of the antibody or its active fragment to detect the human pro-B cell; characterized by using saponin buffer when immunostaining a VpreB molecule in the human pro-B cell.
The invention also provides a diagnostic agent for diagnosing which differentiation stage of B cell acute lymphoblastic leukemia in childhood results from; characterized by containing at least (1) an antibody which recognizes a human pre-B cell receptor and does not recognize a human pro-B cell, (2) an antibody which recognizes a VpreB molecule of a surrogate light chain constituting a human pre-B cell receptor, (3) an antibody which recognizes a xcex5 molecule of a surrogate light chain constituting a human pre-B cell receptor, and (4) an antibody which recognizes a B cell.
The invention also provides a method for diagnosing which differentiation stage of B cell acute lymphoblastic leukemia in childhood results from, by using an antibody which recognizes a human pre-B cell receptor, and does not recognize a human pro-B cell, an antibody which recognizes a VpreB molecule of a surrogate light chain constituting a human pre-B cell receptor, an antibody which recognizes a xcex5 molecule of a surrogate light chain constituting a human pre-B cell receptor, and an antibody which recognizes a B cell.
The present invention will become more fully understood from the detailed description given hereinbelow and the accompanying drawings which are given by way of illustration only, and thus are not to be considered as limiting the present invention.
Further scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.